Dear Patrick, Thank you for the insight!
Regards Kavya On 2024-02-06 00:29, Patrick Shaw Stewart wrote: >> is it logical to say that if the protein is highly charged (either negative >> or positive), it is likely to be more soluble and resist crystallization due >> to electrostatic repulsion? > > Hi Kavyashreem > > The project that I mentioned, where we looked at the crystallization > conditions reported in the PDB, also looked at the areas of amino acids (and > atoms) on the surfaces of proteins and tried to correlate the various groups > with crystallization conditions. > > Positive, negative, charged, polar or hydrophobic groups had very little > effect on the concentration of precipitant (we looked at PEG and ammonium > sulfate) and seemed not to affect the choice of precipitant (looking at all > precipitants). > > The only thing you could say was that the area of all charged and polar > groups as a proportion of the total surface area was roughly constant. If > the charged/polar area was high, the protein was crystallized at slightly > higher protein concentrations, and slightly higher concentrations of > precipitant were used on average. > > But it should be possible to make predictions. DeepMind is probably working > on this right now! > > Best wishes, > > Patrick > > On Mon, Feb 5, 2024 at 3:06 PM kavyashreem <kavyashr...@instem.res.in> wrote: > > Dear all, > > Thank you all for your valuable experiences, inputs and references!! I shall > try them and hope for some good news! Its good to know there are so many > examples of crystallization at such high concentrations. > > A curious question - is it logical to say that if the protein is highly > charged (either negative or positive), it is likely to be more soluble and > resist crystallization due to electrostatic repulsion? Our protein has highly > positively charged surface, although with some small negative patches. > > Following are some of the suggestions indicated: > > 1. Use water (will try) > > 2. Change buffers, pH temperature - (done) > > 3. Seeding (will try) > > 4. Methylating lysines (will try!!) > > 5. Surface entropy reduction (ongoing) > > 6. Optimize constructs (done) > > 7. Different ratios (done) > > 8. Keep concentrating (will try, the problem is yield is low!) > > 9. High salt concentrations (will try) > > 10. Organic solvents (though about it) > > 11. Mutations (ongoing) > > Thank you > > Regards > > Kavya > > On 2024-02-05 15:57, kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules solidified, > we focused on them and expanded with 120mg/ml protein, still there were not > visible precipitates except for the phase separation. This has been a > challenging target so far. We have tried with different constructs, which > unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Thank you > > Regards > > Kavya > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO > COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. > > CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL > AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE > ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER > IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. 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